Gene editing to improve joint function

ABSTRACT

The present invention provides compositions and methods for treating joint disorders that are characterized by an inflammatory component. In some aspects, the compositions and methods are to prevent the progression of osteoarthritis and other arthritides and to treat osteoarthritis and other arthritides in a mammalian joint.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of InternationalApplication No. PCT/US2020/014139, filed on Jan. 17, 2020, which claimspriority to U.S. Provisional Patent Application No. 62/794,340, filed onJan. 18, 2019, U.S. Provisional Patent Application No. 62/894,184, filedon Aug. 30, 2019, and U.S. Provisional Patent Application No.62/914,635, filed on Oct. 14, 2019, each of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

Compositions and Methods for treating synovial joint dysfunction aredescribed herein. In addition, methods for gene-editing synovial cellsand/or synoviocytes, chondrocytes, synovial macrophages, and synovialfibroblasts, and uses of gene-edited synovial cells and/or synoviocytes,chondrocytes, synovial macrophages, and synovial fibroblasts, in thetreatment of diseases such as osteoarthritis are disclosed herein.

BACKGROUND OF THE INVENTION

Treatment of osteoarthritis, degenerative joint disease, and other jointdysfunction is complex and there are few long term options for eithersymptomatic relief or restoring joint function. Osteoarthritis (OA) isthe leading cause of disability due to pain. Neogi, OsteoarthritisCartilage 2013; 21:1145-53. All mammal species are affected: workinganimals, domestic pets, and their owners all suffer OA-relateddiscomfort, pain, and disability, depending on the degree of diseaseprogression.

OA is a complex disease characterized by a progressive course ofdisability. Systemic inflammation is associated with OA and with OAdisease progression. Inflammation is driven by increased levels ofpro-inflammatory cytokines. New methods and compositions to treat thisdisease are acutely needed. Disclosed herein are compositions andmethods useful for treating OA as well as other inflammatory jointdisorders.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The sequence listing contained in the file named“Sequence_Listing_123994-5001-WO.txt” and having a size of 17.1 KBkilobytes, has been submitted electronically herewith via EFS-Web, andthe contents of the txt file are hereby incorporated by reference intheir entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatingjoint disorders that are characterized by an inflammatory component. Insome aspects, the compositions and methods are to prevent theprogression of osteoarthritis and other arthritides and to treatosteoarthritis and other arthritides in a mammalian joint. According toexemplary embodiments, at least a portion of the joint synovial cellsand/or synoviocytes, chondrocytes, synovial macrophages, or synovialfibroblasts are gene-edited to reduce the expression of inflammatorycytokines. In some aspects, at least a portion of the joint synovialcells and/or synoviocytes, chondrocytes, synovial macrophages, orsynovial fibroblasts, are gene-edited to reduce the expression of IL-1α,IL-1β, or both IL-1α, IL-1β.

In some embodiments, the gene-editing causes expression of one or morecytokine and/or growth factor genes to be silenced or reduced in atleast a portion of the cells comprising a mammalian joint. In someaspects, the cells are synovial cells. In some aspects, the cells aresynovial fibroblasts. In some aspects, the cells are synoviocytes. Insome aspects, the cells are chondrocytes. In some aspects, the cells aresynovial macrophages.

In some embodiments, the one or more cytokine and/or growth factor genesis/are selected from the group comprising IL-1α, and IL-1β.

In some embodiments, the gene-editing comprises the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at said one or more cytokine and/or growth factorgenes.

In some embodiments, the gene-editing comprises one or more methodsselected from a CRISPR method, a TALE method, a zinc finger method, anda combination thereof.

In some embodiments, the gene-editing comprises a CRISPR method.

In some embodiments, the CRISPR method is a CRISPR-Cas9 method.

In some embodiments, the gene-editing comprises a TALE method.

In some embodiments, the gene-editing comprises a zinc finger method.

In some embodiments, the gene-editing causes expression of one or morecytokine and/or growth factor genes to be silenced or reduced in atleast a portion of the cells comprising the joint. In some embodiments,the portion of cells edited are synoviocytes. In an aspect, the portionof cells edited are synovial fibroblasts. In some embodiments, theportion of cells edited are synoviocytes. In some embodiments, theportion of cells edited are chondrocytes. In some embodiments, theportion of cells edited are synovial macrophages.

In some embodiments, an adeno-associated virus (AAV) delivery system isused to deliver the gene-editing system. In some embodiments, the AAVdelivery system is injected into a joint.

Some aspects of the present invention provide a pharmaceuticalcomposition for the treatment or prevention of a joint disease orcondition comprising a gene-editing system and a pharmaceuticallyacceptable carrier. In an aspect, the gene-editing system comprises oneor more nucleic acids targeting one or more genetic locus selected fromthe group consisting of IL-1α, IL-1β, TNF-α, IL-6, IL-8, and IL-18.

An embodiment provides a method of treating canine lameness, the methodcomprising administering a gene-editing composition, wherein thecomposition causes expression of IL-1α and IL-1β to be silenced orreduced in a portion of a lame joint's synoviocytes, chondrocytes,synovial macrophages, or synovial fibroblasts.

In some embodiments, the above method further comprises one or morefeatures recited in any of the methods and compositions describedherein.

DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the presently disclosed embodiments.

FIG. 1A illustrates an agarose gel electrophoresis analysis of 100 ngmouse DNA (gBlocks, Integrated DNA Technologies) designed against theMus musculus Il1a and Il1b genes, cleaved by 0.5 μg SpyCas9 (TrueCut™Cas9 protein v2, ThermoFisher Scientific) and 200 ngPhosphorothioate-modified single guide (sg)RNAs targeted against theIl1a gene (#43-46) and Il1b gene (#47-50) in vitro; FIG. 1B illustratesan agarose gel electrophoresis analysis of 100 ng mouse DNA (gBlocks,Integrated DNA Technologies) designed against the Mus musculus Il1a andIl1b genes, cleaved by 0.5 μg SauCas9 (GeneSnipper™ Cas9, BioVision) and200 ng Phosphorothioate-modified guide sgRNAs against the Il1a (#51-53)and IL1b (#54-56) genes.

FIGS. 2A-2D illustrate graphs displaying editing efficiencies of SpyCas9and SauCas9 used with a range of guide RNA's in J774.2 (“J”) and NIH3T3(“N”) cells; FIG. 2A: in vivo cleavage of Il1a, edited with 4×sgRNAs(Spy Cas9) in two separate pools (Pool 1 and 2), across two cell lines,NIH 3T3 (“N”), and J774.2 (“J”); FIG. 2B: in vivo cleavage of Il1b,edited with 4×sgRNAs (Spy Cas9) in two separate pools (Pool 1 and 2),across two cell lines, NIH 3T3 (“N”), and J774.2 (“J”); FIG. 2C: in vivocleavage of Il1a, edited with 3×sgRNAs (Sau Cas9) in two separate pools(Pool 1 and 2), across two cell lines, NIH 3T3 (“N”), and J774.2 (“J”);FIG. 2D: in vivo cleavage of Il1b, edited with 3×sgRNAs (saCas9) in twoseparate pools (Pool 1 and 2), across two cell lines, NIH 3T3 (“N”), andJ774.2 (“J”); editing efficiencies determined using deconvolution ofSanger sequencing traces (ICE tool, Synthego) of each pool.

FIG. 3 illustrates GFP expression measured using the IVIS system. Fluxvalues were based on a region of interest centred on the animal'sinjected knee joint. Data are presented as mean (SD) for four specimensper group.

While the above-identified drawing sets forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, embodiments of the present invention providecompositions and methods for improving joint function and treating jointdisease. In particular embodiments, compositions and methods areprovided to gene-edit synovial fibroblasts, synoviocytes, chondrocytes,or synovial macrophages to reduce expression of inflammatory cytokines,for example, IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-18, one or more matrixmetalloproteinases (MMPs), or one or more component of the NLRP3inflammasome. Embodiments are used for treating osteoarthritis and otherinflammatory joint diseases. Embodiments are further useful for treatingcanine lameness due to osteoarthritis. Embodiments are further usefulfor treating equine lameness due to joint disease. Embodiments are alsouseful for treating post-traumatic arthritis, gout, pseudogout, andother inflammation-mediated or immune-mediated joint diseases.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “ex vivo” refers to an event which involves treating orperforming a procedure on a cell, tissue and/or organ which has beenremoved from a subject's body. Aptly, the cell, tissue and/or organ maybe returned to the subject's body in a method of surgery or treatment.

The term “IL-1” (also referred to herein as “IL1”) refers to thepro-inflammatory cytokine known as interleukin-1, and includes all formsof IL-1, including IL1-α and IL-1β, human and mammalian forms,conservative amino acid substitutions, glycoforms, biosimilars, andvariants thereof. IL-1α and IL-1β bind to the same receptor molecule,which is called type I IL-1 receptor (IL-1RI). There is a third ligandof this receptor: Interleukin 1 receptor antagonist (IL-1Ra), which doesnot activate downstream signaling; therefore, acting as an inhibitor ofIL-1α and IL-1β signaling by competing with them for binding sites ofthe receptor. See, e.g., Dinarello, Blood 117: 3720-32 (2011) and Weberet al., Science Signaling 3(105): cm1, doi:10.1126/scisignal.3105 cm1.IL-1 is described, e.g., in Dinarello, Cytokine Growth Factor Rev.8:253-65 (1997), the disclosures of which are incorporated by referenceherein. For example, the term IL-1 encompasses human, recombinant formsof IL-1.

TABLE 1 Amino acid sequences of interleukins. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1        10         20         30         40         50 recombinantMAKVPDMFED LKNCYSENEE DSSSIDHLSL NQKSFYHVSY GPLHEGCMDQ human IL-1alpha        60         70         80         90        100 (rhIL-1α)SVSLSISETS KTSKLTFKES MVVVATNGKV LKKRRLSLSQ SITDDDLEAI       110        120        130        140        150ANDSEEEIIK PRSAPFSFLS NVKYNFMRII KYEFILNDAL NQSIIRANDQ       160        170        180        190        200YLTAAALHNL DEAVKFDMGA YKSSKDDAKI TVILRISKTQ LYVTAQDEDQ       210        220        230        240        250PVLLKEMPEI PKTITGSETN LLFFWETHGT KNYFTSVAHP NLFIATKQDY       260        270 WVCLAGGPPS ITDFQILENQ A SEQ ID NO: 2        10         20         30         40         50 recombinantMAEVPELASE MMAYYSGNED DLFFEADGPK QMKCSFQDLD LCPLDGGIQL human IL-1beta        60         70         80         90        100 (rhIL-1β)RISDHHYSKG FRQAASVVVA MDKLRKMLVP CPQTFQENDL STFFPFIFEE       110        120        130        140        150EPIFFDTWDN EAYVHDAPVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ       160        170        180        190        200DMEQQVVFSM SFVQGEESND KIPVALGLKE KNLYLSCVLK DDKPTLQLES       210        220        230        240        250VDPKNYPKKK MEKRFVFNKI EINNKLEFES AQFPNWYIST SQAENMPVFL        260GGTKGGQDIT DFTMQFVSS SEQ ID NO: 3        10         20         30         40         50 recombinantMAKVPDLFED LKNCYSENED YSSAIDHLSL NQKSFYDASY GSLHETCTDQ mouse IL-1alpha        60         70         80         90        100 (rmIL-1α)FVSLRTSETS KMSNFTFKES RVTVSATSSN GKILKKRRLS FSETFTEDDL       110        120        130        140        150QSITHDLEET IQPRSAPYTY QSDLRYKLMK LVRQKFVMND SLNQTIYQDV       160        170        180        190        200DKHYLSTTWL NDLQQEVKFD MYAYSSGGDD SKYPVTLKIS DSQLFVSAQG       210        220        230        240        250EDQPVLLKEL PETPKLITGS ETDLIFFWKS INSKNYFTSA AYPELFIATK       260        270 EQSRVHLARG LPSMTDFQIS SEQ ID NO: 4        10         20         30         40         50 recombinantMATVPELNCE MPPFDSDEND LFFEVDGPQK MKGCFQTFDL GCPDESIQLQ mouse IL-1beta        60         70         80         90        100 (rmIL-1β)ISQQHINKSF RQAVSLIVAV EKLWQLPVSF PWTFQDEDMS TFFSFIFEEE       110        120        130        140        150PILCDSWDDD DNLLVCDVPI RQLHYRLRDE QQKSLVLSDP YELKALHLNG       160        170        180        190        200QNINQQVIFS MSFVQGEPSN DKIPVALGLK GKNLYLSCVM KDGTPTLQLE       210        220        230        240        250SVDPKQYPKK KMEKRFVFNK IEVKSKVEFE SAEFPNWYIS TSQAEHKPVF        260LGNNSGQDII DFTMESVSS SEQ ID NO: 5        10         20         30         40         50 recombinantMEICRGLRSH LITLLLFLFH SETICRPSGR KSSKMQAFRI WDVNQKTFYL human IL-1        60         70         80         90        100 receptorRNNQLVAGYL QGPNVNLEEK IDVVPIEPHA LFLGIHGGKM CLSCVKSGDE antagonist       110        120        130        140        150 (rhIL-1Ra)TRLQLEAVNI TDLSENRKQD KRFAFIRSDS GPTTSFESAA CPGWFLCTAM       160        170 EADQPVSLTN MPDEGVMVTK FYFQEDE SEQ ID NO: 6        10         20         30         40         50 recombinantMEICWGPYSH LISLLLILLF HSEAACRPSG KRPCKMQAFR IWDTNQKTFY mouse IL-1        60         70         80         90        100 receptorLRNNQLIAGY LQGPNIKLEE KIDMVPIDLH SVFLGIHGGK LCLSCAKSGD antagonist       110        120        130        140        150 (rmIL-1Ra)DIKLQLEEVN ITDLSKNKEE DKRFTFIRSE KGPTTSFESA ACPGWFLCTT       160        170 LEADRPVSLT NTPEEPLIVT KFYFQEDQ

The term “NLRP3 inflammasome” refers to the multiprotein complexresponsible for the activation of some inflammatory responses. The NMRP3inflammasome promotes the production of functional pro-inflammatorycytokines, for example, IL-1β and IL-18. Core components of the NLRP3inflammasome are NLRP3, ASC (apoptosis-associated speck-like proteincontaining a CARD), and caspase-1, as described by Lee et al., LipidsHealth Dis. 16:271 (2017) and Groslambert and Py, J. Inflamm. Res.11:359-374 (2018).

The terms “matrix metalloproteinase” and “MMP” are defined to be any oneof the members of the matrix metalloproteinase family ofzinc-endopeptidase, for example, as characterized by Fanjul-Fernandez etal., Biochem. Biophys. Acta 1803:3-19 (2010). In the art, family membersare frequently referred to as archetypical MMPs, gelatinases,matrilysins, and/or furin-activatable MMPs. As used herein, the “matrixmetalloproteinase” and “MMP” encompass the entire MMP family, including,but not limited to MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10,MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19,MMP-20, MMP-21, MMP-23, MMP-25, MMP-26, MMP-27 and MMP-28.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one anti-inflammatory compoundin combination with a viral vector functionally engineered to deliver agene-editing nucleic acid as described herein) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a composition or combination of compositions asdescribed herein that is sufficient to effect the intended applicationincluding, but not limited to, disease treatment. A therapeuticallyeffective amount may vary depending upon the intended application (invitro or in vivo), or the subject and disease condition being treated(e.g., the weight, age and gender of the subject), the severity of thedisease condition, or the manner of administration. The term alsoapplies to a dose that will induce a particular response in target cells(e.g., the reduction of platelet adhesion and/or cell migration). Thespecific dose will vary depending on the particular compositions chosen,the dosing regimen to be followed, whether the composition isadministered in combination with other compositions or compounds, timingof administration, the tissue to which it is administered, and thephysical delivery system in which the composition is carried.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, canine,feline, or equine, and includes: (a) preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development or progression; and (c) relieving the disease,i.e., causing regression of the disease and/or relieving one or moredisease symptoms. “Treatment” is also meant to encompass delivery of anagent in order to provide for a pharmacologic effect, even in theabsence of a disease or condition. For example, “treatment” encompassesdelivery of a composition that can elicit an immune response or conferimmunity in the absence of a disease condition, e.g., in the case of avaccine. It is understood that compositions and methods of the presentinvention are applicable to treat all mammals, including, but notlimited to human, canine, feline, equine, and bovine subjects.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” (or synonyms thereof, e.g., “99% identical”) in the context oftwo or more nucleic acids or polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. ClustalW and ClustalX may be used toproduce alignments, Larkin et al., Bioinformatics 23:2947-2948 (2007);Goujon et al., Nucleic Acids Research, 38 Suppl:W 695-9 (2010); and,McWilliam et al., Nucleic Acids Research 41(Web Server issue):W 597-600(2013). One skilled in the art can determine appropriate parameters formaximal alignment by particular alignment software. In certainembodiments, the default parameters of the alignment software are used.

As used herein, the term “variant” encompasses but is not limited toantibodies or fusion proteins which comprise an amino acid sequencewhich differs from the amino acid sequence of a reference antibody byway of one or more substitutions, deletions and/or additions at certainpositions within or adjacent to the amino acid sequence of the referenceantibody. The variant may comprise one or more conservativesubstitutions in its amino acid sequence as compared to the amino acidsequence of a reference antibody. Conservative substitutions mayinvolve, e.g., the substitution of similarly charged or uncharged aminoacids. The variant retains the ability to specifically bind to theantigen of the reference antibody. The term variant also includespegylated antibodies or proteins.

“Carrier” or “vehicle” as used herein refer to carrier materialssuitable for drug administration. Carriers and vehicles useful hereininclude any such materials known in the art, e.g., any liquid, gel,solvent, liquid diluent, solubilizer, surfactant, or the like, which isnontoxic and which does not interact with other components of thecomposition in a deleterious manner.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms that are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” are intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and inert ingredients. The useof such pharmaceutically acceptable carriers or pharmaceuticallyacceptable excipients for active pharmaceutical ingredients is wellknown in the art. Except insofar as any conventional pharmaceuticallyacceptable carrier or pharmaceutically acceptable excipient isincompatible with the active pharmaceutical ingredient, its use in thetherapeutic compositions of the invention is contemplated. Additionalactive pharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The term “pharmaceutically acceptable excipient” is intended to includevehicles and carriers capable of being co-administered with a compoundto facilitate the performance of its intended function. The use of suchmedia for pharmaceutically active substances is well known in the art.Examples of such vehicles and carriers include solutions, solvents,dispersion media, delay agents, emulsions and the like. Any otherconventional carrier suitable for use with the multi-binding compoundsalso falls within the scope of the present disclosure.

As used herein, the term “a”, “an”, or “the” generally is construed tocover both the singular and the plural forms.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat compositions, amounts, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The term “substantially” as used herein can refer to a majority of, ormostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the present invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”

“Joint disease” is defined as measurable abnormalities in the cells ortissues of the joint that could lead to illness, for example, metabolicand molecular derangements triggering anatomical and/or physiologicalchanges in the joint. Including, but not limited to, radiographicdetection of joint space narrowing, subchondral sclerosis, subchondralcysts, and osteophyte formation.

“Joint illness” is defined in human subjects as symptoms that drive thesubject to seek medical intervention, for example, subject reportedpain, stiffness, swelling, or immobility. For non-human mammals, “jointillness” is defined, for example, as lameness, observable changes ingait, weight bearing, allodynia, or exploratory behavior.

As used herein, a sgRNA (single guide RNA) is a RNA, preferably asynthetic RNA, composed of a targeting sequence and scaffold. It is usedto guide Cas9 to a specific genomic locus in genome engineeringexperiments. The sgRNA can be administered or formulated, e.g., as asynthetic RNA, or as a nucleic acid comprising a sequence encoding thegRNA, which is then expressed in the target cells.

As used herein, “Cas9” refers to CRISPR Associated Protein; the Cas9nuclease is the active enzyme for the Type II CRISPR system. “nCas9”refers to a Cas9 that has one of the two nuclease domains inactivated,i.e., either the RuvC or HNH domain. nCas9 is capable of cleaving onlyone strand of target DNA (a “nickase”).

As used herein, “PAM” refers to a Protospacer Adjacent Motif and isnecessary for Cas9 to bind target DNA, and immediately follows thetarget sequence. The Cas9 can be administered or formulated, e.g., as aprotein (e.g., a recombinant protein), or as a nucleic acid comprising asequence encoding the Cas9 protein, which is then expressed in thetarget cells.

A subject treated by any of the methods or compositions described hereincan be of any age and can be an adult, infant or child. In some cases,the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 years old, or within arange therein (e.g., without limitation, between 2 and 20 years old,between 20 and 40 years old, or between 40 and 90 years old). Thesubject can be a human or non-human subject. A particular class ofsubjects that can benefit from the compositions and methods of thepresent disclosure include subjects over the age of 40, 50, or 60 years.Another class of subjects that can benefit from the compositions andmethods of the present disclosure are subjects that have arthritis(e.g., osteoarthritis).

Any of the compositions disclosed herein can be administered to anon-human subject, such as a laboratory or farm animal. Non-limitingexamples of a non-human subject include laboratory or research animals,pets, wild or domestic animals, farm animals, etc., e.g., a dog, a goat,a guinea pig, a hamster, a mouse, a pig, a non-human primate (e.g., agorilla, an ape, an orangutan, a lemur, a baboon, etc.), a rat, a sheep,a horse, a cow, or the like.

The present invention provides compositions useful for treating jointdisorders with an inflammatory component. In some aspects, thecompositions are useful to prevent the progression of osteoarthritis andto treat osteoarthritis in a mammalian joint.

In some aspects, the pharmaceutical composition comprises a gene-editingsystem, wherein the gene-editing system causes expression the at leastone genetic locus related to joint function to be silenced or reduced inat least a portion of the cells comprising the joint.

In an aspect, the pharmaceutical composition comprises a gene-editingsystem, wherein the gene-editing system targets one or more of IL-1α,and IL-1β. In some aspects, the pharmaceutical composition comprises agene-editing system, wherein the gene-editing system targets one or moreof TNF-α, IL-6, IL-8, IL-18, a matrix metalloproteinase (MMP), orcomponents of the NLRP3 inflammasome.

In some aspects, the pharmaceutical composition comprises a gene-editingsystem, wherein the gene-editing comprises the use of a programmablenuclease that mediates the generation of a double-strand orsingle-strand break at the at least one locus related to joint function.In some embodiments, the gene-editing system reduces the gene expressionof the targeted locus or targeted loci. In some embodiments, the atleast one locus related to joint tissue is silenced or reduced in atleast a portion of the cells comprising the joint.

In some aspects, the cells comprising the joint are synoviocytes. Insome aspects, the cells are synovial macrophages. In some aspects, thecells are synovial fibroblasts. In some aspects at least a portion ofthe synoviocytes are edited. In some aspects, the cells comprising thejoint are chondrocytes.

In an aspect, the pharmaceutical composition targets the one or morecytokine and/or growth factor genes is/are selected from the groupcomprising IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-18, a matrixmetalloproteinase (MMP), or a component of the NLRP3 inflammasome. Insome embodiments, the component of the NLRP3 inflammasome comprisesNLRP3, ASC (apoptosis-associated speck-like protein containing a CARD),caspase-1, and combinations thereof.

Pharmaceutical compositions are also provided, wherein the gene-editingcauses expression of one or more cytokine and/or growth factor genes tobe enhanced in at least a portion of the cells comprising the joint, thecytokine and/or growth factor gene(s) being selected from the groupcomprising IL-1Ra, TIMP-1, TIMP-2, TIMP-3, TIMP-4, and combinationsthereof.

In some embodiments, the pharmaceutical composition provides forgene-editing, wherein the gene-editing comprises the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at said one or more cytokine and/or growth factorgenes. In some embodiments, the gene-editing comprises one or moremethods selected from a CRISPR method, a TALE method, a zinc fingermethod, and a combination thereof.

In an aspect, the gene-editing comprises a CRISPR method. In yet otheraspects, the CRISPR method is a CRISPR-Cas9 method. In some aspects, theCas9 is mutated to enhance function.

Animal Models of Osteoarthritis

Several animal models for osteoarthritis are known to the art. Exemplarynonlimiting animal models are summarized; however, it is understood thatvarious models may be used. Many different species of animals are usedto mimic OA, for example, studies have been conducted on mice, rats,rabbits, guinea pigs, dogs, pigs, horses, and even other animals. See,e.g., Kuyinu et al., J Orthop Surg Res. 11:19 (2016) (hereinafter“Kuyinu; 2016”).

It is understood that the various methods for inducing OA may be used inany mammal. In the mouse, spontaneous, chemically induced, surgicallyinduced, and non-invasive induction are commonly used. E.g., Kuyinu,2016; Bapat et al., Clin Transl Med. 7:36 (2018) (hereinafter “Bapat,2018”); and Poulet, Curr Rheumatol Rep 18:40 (2016). In the horse,osteochondral fragment-exercise model, chemical induction, traumaticinduction, and induction through overuse are commonly used. In sheep,surgical induction is most common; in the guinea pig, surgicalinduction, chemical induction, and spontaneous (Durkin Hartley) methodsare frequently used. E.g. Bapat, 2018.

The destabilized medial meniscus (DMM) is frequently used in mice tomodel posttraumatic osteoarthritis, e.g. Culley et al., Methods MolBiol.1226:143-73 (2015). The DMM model mimics clinical meniscal injury,a known predisposing factor for the development of human OA, and permitsthe study of structural and biological changes over the course of thedisease. Mice are an attractive model organism, because mouse strainswith defined genetic backgrounds may be used. Additionally, knock-out orother genetically manipulated mouse strains may be used to evaluate theimportance of various molecular pathways in the response to various OAtreatment modalities and regimens. For example, STR/ort mice havefeatures that make the strain particularly susceptible to developing OA,including, increased levels of the inflammatory cytokine IL1β, Bapat etal., Clin Transl Med. 7:36 (2018). These mice commonly develop OA inknee, ankle, elbow, and temporo-mandibular joints, Jaeger et al.,Osteoarthritis Cartilage 16:607-614 (2008). Other useful mutant strainsof mice are known to the skilled artisan, for example, Col9a1(−/−) mice,Allen et al., Arthritis Rheum, 60:2684-2693 (2009).

Another commonly used surgical model for OA is anterior cruciateligament transection (ACLT) model. Little and Hunter, Nat RevRheumatol., 9(8):485-497 (2013). The subject's ACL is surgicallytransected causing joint destabilization. The anterior drawer test withthe joint flexed is used to confirm that transection of the ligament hasoccurred. In some cases, other ligaments such as the posterior cruciateligament, medial collateral ligament, lateral collateral ligament,and/or either meniscus may be transected. As with the DMM model, avariety of mouse strains may be used to investigate various molecularpathways.

Depending on the technical objective, animals of varying size may beselected for use. Rodents are useful because of the short time neededfor skeletal maturity and consequently shorter time to develop OAfollowing surgical or other technique to induce OA. Larger animals areparticularly useful to evaluate therapeutic interventions. The anatomyin larger animals is very similar to humans; for example, in dogs thecartilage thickness is less than about half the thickness of humans;this striking similarity is exemplary of why such cartilage degenerationand osteochondral defects studies are much more useful in large animalmodels. E.g. McCoy, Vet. Pathol., 52:803-18 (2015); and, Pelletier etal., Therapy, 7:621-34(2010).

Gene-Editing Processes

Overview: Compositions to gene-edit Synovial Cells

Embodiments of the present invention are directed to methods forgene-editing synovial cells (synoviocytes), the methods comprising oneor more steps of gene-editing at least a portion of the synoviocytes ina joint to treat osteoarthritis or other joint disorder. As used herein,“gene-editing,” “gene editing,” and “genome editing” refer to a type ofgenetic modification in which DNA is permanently modified in the genomeof a cell, e.g., DNA is inserted, deleted, modified or replaced withinthe cell's genome. In some embodiments, gene-editing causes theexpression of a DNA sequence to be silenced (sometimes referred to as agene knockout) or inhibited/reduced (sometimes referred to as a geneknockdown). In other embodiments, gene-editing causes the expression ofa DNA sequence to be enhanced (e.g., by causing over-expression). Inaccordance with embodiments of the present invention, gene-editingtechnology is used to reduce the expression or silence pro-inflammatorygenes and/or to enhance the expression of regenerative genes.

Interleukins

According to additional embodiments, gene-editing methods of the presentinvention may be used to increase the expression of certaininterleukins, such as one or more of IL-1α, IL-1β, IL-4, IL-6, IL-8,IL-9, IL-10, IL-13, IL-18, and TNF-α. Certain interleukins have beendemonstrated to augment inflammatory responses in joint tissue and arelinked to disease progression.

Expression Constructs

Expression constructs encoding one or both of guide RNAs and/or Cas9editing enzymes can be administered in any effective carrier, e.g., anyformulation or composition capable of effectively delivering thecomponent gene to cells in vivo. Approaches include, for example,electroporation and/or insertion of the gene in viral vectors, includingrecombinant retroviruses, adenovirus, adeno-associated virus,lentivirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNAcan be delivered naked or with the help of, for example, cationicliposomes (lipofectamine) or derivatized (e.g., antibody conjugated),polylysine conjugates, gramacidin S, artificial viral envelopes or othersuch intracellular carriers, as well as direct injection of the geneconstruct or CaPO4 precipitation carried out in vivo.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g., a cDNA.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells thathave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo, particularly into humans. These vectors provide efficient deliveryof genes into cells. In some instances, the transferred nucleic acidsare stably integrated into the chromosomal DNA of the host. In otherinstances, particularly for adeno-associated virus vectors, stableintegration into the host DNA may be a rare event, resulting intoepisomal expression of the transgene and transient expression of thetransgene.

The development of specialized cell lines (termed “packaging cells”)which produce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, Blood 76:271 (1990)). A replication defectiveretrovirus can be packaged into virions, which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Ausubel, et al.,eds., Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989), Sections 9.10-9.14, and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see, e.g.,Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988)Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc.Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl.Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci.USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573, each of which is incorporated by referenceherein in its entirety for all purposes).

Another viral gene delivery system useful in the present methodsutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated, such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al.,BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434(1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitableadenoviral vectors may be derived from any strain of adenovirus (e.g.,Ad2, Ad3, Ad5, or Ad7 etc.), including Adenovirus serotypes from otherspecies (e.g., mouse, dog, human, etc.) that are known to those skilledin the art. The virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis in situ,where introduced DNA becomes integrated into the host genome (e.g.,retroviral DNA). Moreover, the carrying capacity of the adenoviralgenome for foreign DNA is large (up to 8 kilobases) relative to othergene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J.Virol. 57:267 (1986).

Helper-dependent (HDAd) vectors can also be produced with all adenoviralsequences deleted except the origin of DNA replication at each end ofthe viral DNA along with packaging signal at 5-prime end of the genomedownstream of the left packaging signal. HDAd vectors are constructedand propagated in the presence of a replication-competent helperadenovirus that provides the required early and late proteins necessaryfor replication.

Yet another viral vector system useful for delivery of nucleic acids isthe adeno-associated virus (AAV). Adeno-associated virus is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal., Curr. Topics in Micro. and Immunol. 158:97-129 (1992). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992);Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al.,J. Virol. 62:1963-1973 (1989). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984);Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford etal., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol.51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790(1993). The identification of Staphylococcus aureus (SaCas9) and othersmaller Cas9 enzymes that can be packaged into adeno-associated viral(AAV) vectors that are highly stable and effective in vivo, easilyproduced, approved by FDA, and tested in multiple clinical trials, pavesnew avenues for therapeutic gene editing.

In some embodiments, nucleic acids encoding a CRISPR IL-1α or IL-1β geneediting complex (e.g., Cas9 or gRNA) are entrapped in liposomes bearingpositive charges on their surface (e.g., lipofectins), which can betagged with antibodies against cell surface antigens of the targetcells. These delivery vehicles can also be used to deliver Cas9protein/gRNA complexes.

In clinical settings, the gene delivery systems for the nucleic acidsencoding a CRISPR IL-1α or IL-1β gene editing complex can be introducedinto a subject by any of a number of methods, each of which is familiarin the art. For instance, a pharmaceutical preparation of the genedelivery system can be introduced systemically, e.g., by intravenousinjection, and specific transduction of the protein in the target cellswill occur predominantly from specificity of transfection, provided bythe gene delivery vehicle, cell-type or tissue-type expression due tothe transcriptional regulatory sequences controlling expression of thereceptor gene, or a combination thereof. In other embodiments, initialdelivery of the nucleic acids encoding a CRISPR IL-1α or IL-1β geneediting complex is more limited, with introduction into the subjectbeing quite localized. For example, the nucleic acids encoding a CRISPRIL-1α or IL-1β gene editing complex can be introduced by intra-articularinjection into a joint exhibiting joint disease (e.g., osteoarthritis).In some embodiments, the nucleic acids encoding a CRISPR IL-1α or IL-1βgene editing complex are administered during or after surgery; in someembodiments, a controlled-release hydrogel comprising the nucleic acidsencoding a CRISPR IL-1α or IL-1β gene editing complex is administered atthe conclusion of surgery before closure to prevent reduce or eliminateosteoarthritis by providing a steady dose of the nucleic acids encodinga CRISPR IL-1α or IL-1β gene editing complex over time.

A pharmaceutical preparation of the nucleic acids encoding a CRISPRIL-1α or IL-1β gene editing complex can consist essentially of the genedelivery system (e.g., viral vector(s)) in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isembedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g., adeno-associated viralvectors, the pharmaceutical preparation can comprise one or more cells,which produce the gene delivery system.

Preferably, the CRISPR IL-1α or IL-1β editing complex is specific, i.e.,induces genomic alterations preferentially at the target site (IL-1α orIL-1β), and does not induce alterations at other sites, or only rarelyinduces alterations at other sites. In certain embodiments, the CRISPRIL-1α or IL-1β editing complex has an editing efficiency of at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 99%. In certain embodiments, the sequence of a guideRNA (e.g., a single guide RNA) may be modified to increase editingefficiency and/or reduce off-target effects. In certain embodiments, thesequence of a guide RNA may vary from the target sequence by about 1base, about 2 bases, about 3 bases, about 4 bases, about 5 bases, about5 bases, about 6 bases, about 7 bases, about 8 bases, about 9 bases,about 10 bases, about 15 bases, or greater than about 15 bases. Incertain embodiments, the sequence of a guide RNA may vary from thetarget sequence by about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, or greater than about 20%. As used herein, variationform a target sequence may refer to the degree of complementarity.

Gene Editing Methods

As discussed above, embodiments of the present invention providecompositions and methods to treat joint disorders, wherein a portion ofthe joint cells are genetically modified via gene-editing to treat ajoint disorder. Embodiments of the present invention embrace geneticediting through nucleotide insertion (RNA or DNA), or recombinantprotein insertion, into a population of synoviocytes for both promotionof the expression of one or more proteins and inhibition of theexpression of one or more proteins, as well as combinations thereof.Embodiments of the present invention also provide methods for deliveringgene-editing compositions to joint cells, and in particular deliveringgene-editing compositions to synoviocytes. There are severalgene-editing technologies that may be used to genetically modify jointcells, which are suitable for use in accordance with the presentinvention.

In some embodiments, a method of genetically modifying joint cellsincludes the step of stable incorporation of genes for production of oneor more proteins. In an embodiment, a method of genetically modifying aportion of a joint's synoviocytes includes the step of retroviraltransduction. In an embodiment, a method of genetically modifying aportion of a joint's synoviocytes includes the step of lentiviraltransduction. Lentiviral transduction systems are known in the art andare described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006,103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75;Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Pat. No.6,627,442, the disclosures of each of which are incorporated byreference herein. In an embodiment, a method of genetically modifying aportion of a joint's synoviocytes includes the step of gamma-retroviraltransduction. Gamma-retroviral transduction systems are known in the artand are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996,9.9.1-9.9.16, the disclosure of which is incorporated by referenceherein. In an embodiment, a method of genetically modifying a portion ofa joint's synoviocytes includes the step of transposon-mediated genetransfer. Transposon-mediated gene transfer systems are known in the artand include systems wherein the transposase is provided as DNAexpression vector or as an expressible RNA or a protein such thatlong-term expression of the transposase does not occur in the transgeniccells, for example, a transposase provided as an mRNA (e.g., an mRNAcomprising a cap and poly-A tail). Suitable transposon-mediated genetransfer systems, including the salmonid-type Tel-like transposase (SBor Sleeping Beauty transposase), such as SB10, SB11, and SB100×, andengineered enzymes with increased enzymatic activity, are described in,e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No.6,489,458, the disclosures of each of which are incorporated byreference herein.

In some aspects, viral vectors or systems are used to introduce agene-editing system into cells comprising a joint. In some aspects, thecells are synovial fibroblasts. In some aspects, the viral vectors arean AAV vector. In some aspects, the AAV vector comprises a serotypeselected from the group consisting of: AAV1, AAV1(Y705+731F+T492V),AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV4, AAV5,AAV5(Y436+693+719F), AAV6, AAV6 (VP3 variant Y705F/Y731F/T492V),AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9 (VP3 variant Y731F),AAV10(Y733F), AAV-ShH10, and AAV-DJ/8. In some aspects, the AAV vectorcomprises a serotype selected from the group consisting of: AAV1, AAV5,AAV6, AAV6 (Y705F/Y731F/T492V), AAV8, AAV9, and AAV9 (Y731F).

In some aspects, the viral vector is a lentivirus. In an aspect, thelentivirus is selected from the group consisting of: humanimmunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simianimmunodeficiency virus (SIV), feline immunodeficiency virus (FIV),bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV),equine infectious anemia virus (EIAV), and caprine arthritisencephalitis virus (CAEV).

In an embodiment, a method of genetically modifying a portion of ajoint's synoviocytes includes the step of stable incorporation of genesfor production or inhibition (e.g., silencing) of one or more proteins.In an embodiment, a method of genetically modifying a portion of ajoint's synoviocytes includes the step of liposomal transfection.Liposomal transfection methods, such as methods that employ a 1:1 (w/w)liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are knownin the art and are described in Rose, et al., Biotechniques 1991, 10,520-525 and Feigner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84,7413-7417 and in U.S. Pat. Nos. 5,279,833; 5,908,635; 6,056,938;6,110,490; 6,534,484; and 7,687,070, the disclosures of each of whichare incorporated by reference herein. In an embodiment, a method ofgenetically modifying a portion of a joint's synoviocytes includes thestep of transfection using methods described in U.S. Pat. Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; thedisclosures of each of which are incorporated by reference herein.

According to an embodiment, the gene-editing process may comprise theuse of a programmable nuclease that mediates the generation of adouble-strand or single-strand break at one or more immune checkpointgenes. Such programmable nucleases enable precise genome editing byintroducing breaks at specific genomic loci, i.e., they rely on therecognition of a specific DNA sequence within the genome to target anuclease domain to this location and mediate the generation of adouble-strand break at the target sequence. A double-strand break in theDNA subsequently recruits endogenous repair machinery to the break siteto mediate genome editing by either non-homologous end-joining (NHEJ) orhomology-directed repair (HDR). Thus, the repair of the break can resultin the introduction of insertion/deletion mutations that disrupt (e.g.,silence, repress, or enhance) the target gene product.

Major classes of nucleases that have been developed to enablesite-specific genomic editing include zinc finger nucleases (ZFNs),transcription activator-like nucleases (TALENs), and CRISPR-associatednucleases (e.g., CRISPR-Cas9). These nuclease systems can be broadlyclassified into two categories based on their mode of DNA recognition:ZFNs and TALENs achieve specific DNA binding via protein-DNAinteractions, whereas CRISPR systems, such as Cas9, are targeted tospecific DNA sequences by a short RNA guide molecule that base-pairsdirectly with the target DNA and by protein-DNA interactions. See, e.g.,Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that may be used inaccordance with the methods of the present invention include CRISPRmethods, TALE methods, and ZFN methods, which are described in moredetail below.

CRISPR Methods

A pharmaceutical composition for the treatment or prevention of a jointdisease or condition comprising a gene-editing system, wherein saidgene-editing system targets at least one locus related to jointfunction, wherein the gene-editing at least a portion of a joint'ssynoviocytes by a CRISPR method (e.g., CRISPR-Cas9, CRISPR-Cas13a, orCRISPR/Cpf1 (also known as CRISPR-Cas12a). According to particularembodiments, the use of a CRISPR method to gene-edit joint synoviocytescauses expression of one or more immune checkpoint genes to be silencedor reduced in at least a portion of the joint's synoviocytes.

CRISPR stands for “Clustered Regularly Interspaced Short PalindromicRepeats.” A method of using a CRISPR system for gene editing is alsoreferred to herein as a CRISPR method. There are three types of CRISPRsystems which incorporate RNAs and Cas proteins, and which may be usedin accordance with the present invention: Types II, V, and VI. The TypeII CRISPR (exemplified by Cas9) is one of the most well-characterizedsystems.

CRISPR technology was adapted from the natural defense mechanisms ofbacteria and archaea (the domain of single-celled microorganisms). Theseorganisms use CRISPR-derived RNA and various Cas proteins, includingCas9, to foil attacks by viruses and other foreign bodies by chopping upand destroying the DNA, or RNA, of a foreign invader. A CRISPR is aspecialized region of DNA with two distinct characteristics: thepresence of nucleotide repeats and spacers. Repeated sequences ofnucleotides are distributed throughout a CRISPR region with shortsegments of foreign DNA (spacers) interspersed among the repeatedsequences. In the type II CRISPR-Cas system, spacers are integratedwithin the CRISPR genomic loci and transcribed and processed into shortCRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNAs(tracrRNAs) and direct sequence-specific cleavage and silencing ofpathogenic DNA by Cas proteins. Target recognition by the Cas9 proteinrequires a “seed” sequence within the crRNA and a conserveddinucleotide-containing protospacer adjacent motif (PAM) sequenceupstream of the crRNA-binding region. The CRISPR-Cas system can therebybe retargeted to cleave virtually any DNA sequence by redesigning thecrRNA. The crRNA and tracrRNA in the native system can be simplifiedinto a single guide RNA (sgRNA) of approximately 100 nucleotides for usein genetic engineering. The CRISPR-Cas system is directly portable tohuman cells by co-delivery of plasmids expressing the Cas9 endo-nucleaseand the necessary crRNA and tracrRNA (or sgRNA)components. Differentvariants of Cas proteins may be used to reduce targeting limitations(e.g., orthologs of Cas9, such as Cpf1).

Non-limiting examples of genes that may be silenced or inhibited bypermanently gene-editing synoviocytes via a CRISPR method include IL-1α,IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

Non-limiting examples of genes that may be enhanced by permanentlygene-editing synoviocytes via a CRISPR method include IL-1α, IL-1β,IL-4, IL-9, IL-10, IL-13, and TNF-α.

Examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a CRISPR method, and which maybe used in accordance with embodiments of the present invention, aredescribed in U.S. Pat. Nos. 8,697,359; 8,993,233; 8,795,965; 8,771,945;8,889,356; 8,865,406; 8,999,641; 8,945,839; 8,932,814; 8,871,445;8,906,616; and 8,895,308, which are incorporated by reference herein.Resources for carrying out CRISPR methods, such as plasmids forexpressing CRISPR-Cas9 and CRISPR-Cpf1, are commercially available fromcompanies such as GenScript.

In an embodiment, genetic modifications of at least a portion of ajoint's synoviocytes, as described herein, may be performed using theCRISPR-Cpf1 system as described in U.S. Pat. No. 9,790,490, thedisclosure of which is incorporated by reference herein.

In an embodiment, genetic modifications of at least a portion of ajoint's synoviocytes, as described herein, may be performed using aCRISPR-Cas system comprising single vector systems as described in U.S.Pat. No. 9,907,863, the disclosure of which is incorporated by referenceherein. TALE Methods

A pharmaceutical composition for the treatment or prevention of a jointdisease or condition comprising a gene-editing system, wherein saidgene-editing system targets at least one locus related to jointfunction, wherein the method further comprises gene-editing at least aportion of joint synoviocytes by a TALE method. According to particularembodiments, the use of a TALE method to target at least one locusrelated to joint function, wherein the gene-editing at least a portionof a joint's synoviocytes. Alternatively, the use of a TALE methodduring to target at least one locus related to joint function, whereinthe gene-editing at least a portion of a joint's synoviocytes to causeexpression of at least one locus related to joint function genes to beenhanced in at least a portion of the joint synoviocytes.

TALE stands for “Transcription Activator-Like Effector” proteins, whichinclude TALENs (“Transcription Activator-Like Effector Nucleases”). Amethod of using a TALE system for gene editing may also be referred toherein as a TALE method. TALEs are naturally occurring proteins from theplant pathogenic bacteria genus Xanthomonas, and contain DNA-bindingdomains composed of a series of 33-35-amino-acid repeat domains thateach recognizes a single base pair. TALE specificity is determined bytwo hypervariable amino acids that are known as the repeat-variabledi-residues (RVDs). Modular TALE repeats are linked together torecognize contiguous DNA sequences. A specific RVD in the DNA-bindingdomain recognizes a base in the target locus, providing a structuralfeature to assemble predictable DNA-binding domains. The DNA bindingdomains of a TALE are fused to the catalytic domain of a type IIS FokIendonuclease to make a targetable TALE nuclease. To induce site-specificmutation, two individual TALEN arms, separated by a 14-20 base pairspacer region, bring FokI monomers in close proximity to dimerize andproduce a targeted double-strand break.

Several large, systematic studies utilizing various assembly methodshave indicated that TALE repeats can be combined to recognize virtuallyany user-defined sequence. Custom-designed TALE arrays are alsocommercially available through Cellectis Bioresearch (Paris, France),Transposagen Biopharmaceuticals (Lexington, Ky., USA), and LifeTechnologies (Grand Island, N.Y., USA). TALE and TALEN methods suitablefor use in the present invention are described in U.S. PatentApplication Publication Nos. US 2011/0201118 A1; US 2013/0117869 A1; US2013/0315884 A1; US 2015/0203871 A1 and US 2016/0120906 A1, thedisclosures of which are incorporated by reference herein.

Non-limiting examples of genes that may be silenced or inhibited bypermanently gene-editing synoviocytes via a TALE method include IL-1α,IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α.

Non-limiting examples of genes that may be enhanced by permanentlygene-editing synoviocytes via a TALE method include IL-1α, IL-1β, IL-4,IL-9, IL-10, IL-13, and TNF-α.

Examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a TALE method, and which may beused in accordance with embodiments of the present invention, aredescribed in U.S. Pat. No. 8,586,526, which is incorporated by referenceherein.

Zinc Finger Methods

A pharmaceutical composition for the treatment or prevention of a jointdisease or condition comprising a gene-editing system, wherein saidgene-editing system targets at least one locus related to jointfunction, wherein the method further comprises gene-editing at least aportion of joint synoviocytes by a zinc finger or zinc finger nucleasemethod. According to particular embodiments, the use of a zinc fingermethod to target at least one locus related to joint function, whereinthe gene-editing at least a portion of a joint's synoviocytes.Alternatively, the use of a zinc finger method during to target at leastone locus related to joint function, wherein the gene-editing at least aportion of a joint's synoviocytes to cause expression of at least onelocus related to joint function genes to be enhanced in at least aportion of the joint synoviocytes.

An individual zinc finger contains approximately 30 amino acids in aconserved ββα configuration. Several amino acids on the surface of theα-helix typically contact 3 bp in the major groove of DNA, with varyinglevels of selectivity. Zinc fingers have two protein domains. The firstdomain is the DNA binding domain, which includes eukaryotictranscription factors and contain the zinc finger. The second domain isthe nuclease domain, which includes the FokI restriction enzyme and isresponsible for the catalytic cleavage of DNA.

The DNA-binding domains of individual ZFNs typically contain betweenthree and six individual zinc finger repeats and can each recognizebetween 9 and 18 base pairs. If the zinc finger domains are specific fortheir intended target site then even a pair of 3-finger ZFNs thatrecognize a total of 18 base pairs can, in theory, target a single locusin a mammalian genome. One method to generate new zinc-finger arrays isto combine smaller zinc-finger “modules” of known specificity. The mostcommon modular assembly process involves combining three separate zincfingers that can each recognize a 3 base pair DNA sequence to generate a3-finger array that can recognize a 9 base pair target site.Alternatively, selection-based approaches, such as oligomerized poolengineering (OPEN) can be used to select for new zinc-finger arrays fromrandomized libraries that take into consideration context-dependentinteractions between neighboring fingers. Engineered zinc fingers areavailable commercially; Sangamo Biosciences (Richmond, Calif., USA) hasdeveloped a propriety platform (CompoZr®) for zinc-finger constructionin partnership with Sigma-Aldrich (St. Louis, Mo., USA).

Non-limiting examples of genes that may be silenced or inhibited bypermanently gene-editing synoviocytes via a zinc finger method includeIL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, TNF-α, IL-6, IL-8, IL-18, amatrix metalloproteinase (MMP), or a component of the NLRP3inflammasome. In some embodiments, the component of the NLRP3inflammasome comprises NLRP3, ASC (apoptosis-associated speck-likeprotein containing a CARD), caspase-1, and combinations thereof.

Non-limiting examples of genes that may be enhanced by permanentlygene-editing synoviocytes via a zinc finger method include groupcomprising IL-1Ra, TIMP-1, TIMP-2, TIMP-3, TIMP-4, and combinationsthereof. In an aspect, the invention provides compositions forup-regulation of anti-inflammatory cytokines.

Examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a zinc finger method, which maybe used in accordance with embodiments of the present invention, aredescribed in U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136,6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215,7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and6,479,626, which are incorporated by reference herein.

In some aspects, cells may be gene-edited ex vivo, wherein thegene-editing targets one or more anti-inflammatory cytokine locus. Insome aspects, the cells are non-synovial cells. In some aspects, thecells are mesenchymal stem cells. In some aspect, the cells aremacrophages. In some aspects, the present invention provides for apharmaceutical composition for the treatment or prevention of a jointdisease or condition comprising a population of gene-edited cells,wherein said gene-edited cells are edited by a gene-editing systemtargeting at least one locus related to joint function. In an aspect,the population of gene-edited cells are injected into a synovial joint.

Other examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a zinc finger method, which maybe used in accordance with embodiments of the present invention, aredescribed in Beane, et al., Mol. Therapy, 2015, 23 1380-1390, thedisclosure of which is incorporated by reference herein.

Methods of Treating Osteoarthritis and Other Diseases

The compositions and methods described herein can be used in a methodfor treating diseases. In an embodiment, they are for use in treatinginflammatory joint disorders. They may also be used in treating otherdisorders as described herein and in the following paragraphs. In anaspect, the compositions and methods are used to treat osteoarthritis(OA).

In some embodiments, the present disclosure provides a method for thetreatment or prevention of a joint disease or condition the methodcomprising introducing a gene-editing system, wherein the gene-editingsystem targets at least one locus related to joint function. In someembodiments, the joint disease is osteoarthritis. In an aspect, themethod is used to treat a canine with osteoarthritis. In another aspect,the method is used to treat a mammal with degenerative joint disease. Insome aspects, the method is used to treat a canine or an equine with ajoint disease. In some aspects, the method is used to treatosteoarthritis, post-traumatic arthritis, post-infectious arthritis,rheumatoid arthritis, gout, pseudogout, auto-immune mediatedarthritides, inflammatory arthritides, inflammation-mediated andimmune-mediated diseases of joints.

In some embodiments, the method further comprises gene-editing a portionof a the joint synoviocytes to reduce or silence the expression of oneor more of IL-1α, IL-1β, IL-4, IL-9, IL-10, IL-13, and TNF-α. In anaspect, the method further comprises gene-editing a portion of a thejoint synoviocytes to reduce or silence the expression of one or more ofIL-1α, IL-1β.

In an aspect, the method further comprises gene-editing, wherein thegene-editing comprises one or more methods selected from a CRISPRmethod, a TALE method, a zinc finger method, and a combination thereof.

In some aspects, the method further comprises delivering thegene-editing using an AAV vector, a lentiviral vector, or a retroviralvector. In a preferred embodiment, the method further comprisesdelivering the gene-editing using AAV1, AAV1(Y705+731F+T492V),AAV2(Y444+500+730F+T491V), AAV3(Y705+73IF), AAV5, AAV5(Y436+693+719F),AAV6, AAV6 (VP3 variant Y705F/Y731F/T492V), AAV-7m8, AAV8, AAV8(Y733F),AAV9, AAV9 (VP3 variant Y731F), AAV10(Y733F), and AAV-ShH10. In someaspects, the AAV vector comprises a serotype selected from the groupconsisting of: AAV1, AAV5, AAV6, AAV6 (Y705F/Y731F/T492V), AAV8, AAV9,and AAV9 (Y731F).

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the use of pharmaceuticalcompositions comprising CRISPR gene (e.g., IL-1α and/or IL-1β) editingcomplexes as an active ingredient.

Depending on the method/route of administration, pharmaceutical dosageforms come in several types. These include many kinds of liquid, solid,and semisolid dosage forms. Common pharmaceutical dosage forms includepill, tablet, or capsule, drink or syrup, and natural or herbal formsuch as plant or food of sorts, among many others. Notably, the route ofadministration (ROA) for drug delivery is dependent on the dosage formof the substance in question. A liquid pharmaceutical dosage form is theliquid form of a dose of a chemical compound used as a drug ormedication intended for administration or consumption.

In one embodiment, a composition of the present disclosure can bedelivered to a subject subcutaneously (e.g., intra-articular injection),dermally (e.g., transdermally via patch), and/or via implant. Exemplarypharmaceutical dosage forms include, e.g., pills, osmotic deliverysystems, elixirs, emulsions, hydrogels, suspensions, syrups, capsules,tablets, orally dissolving tablets (ODTs), gel capsules, thin films,adhesive topical patches, lollipops, lozenges, chewing gum, dry powderinhalers (DPIs), vaporizers, nebulizers, metered dose inhalers (MDIs),ointments, transdermal patches, intradermal implants, subcutaneousimplants, and transdermal implants.

As used herein, “dermal delivery” or “dermal administration” can referto a route of administration wherein the pharmaceutical dosage form istaken to, or through, the dermis (i.e., layer of skin between theepidermis (with which it makes up the cutis) and subcutaneous tissues).“Subcutaneous delivery” can refer to a route of administration whereinthe pharmaceutical dosage form is to or beneath the subcutaneous tissuelayer.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker. N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Therapeutic compounds that are or include nucleic acids can beadministered by any method suitable for administration of nucleic acidagents, such as a DNA vaccine. These methods include gene guns, bioinjectors, and skin patches as well as needle-free methods such as themicro-particle DNA vaccine technology disclosed in U.S. Pat. No.6,194,389, and the mammalian transdermal needle-free vaccination withpowder-form vaccine as disclosed in U.S. Pat. No. 6,168,587.Additionally, intranasal delivery is possible, as described in, interalia, Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10(1998). Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) andmicroencapsulation can also be used. Biodegradable targetablemicroparticle delivery systems can also be used (e.g., as described inU.S. Pat. No. 6,471,996).

Therapeutic compounds can be prepared with carriers that will protectthe therapeutic compounds against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as collagen, ethylene vinyl acetate,polyanhydrides (e.g.,poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix,fatty acid dimer-sebacic acid (FAD-SA) copolymer,poly(lactide-co-glycolide)), polyglycolic acid, collagen,polyorthoesters, polyethyleneglycol-coated liposomes, and polylacticacid. Such formulations can be prepared using standard techniques, orobtained commercially, e.g., from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to selected cells with monoclonal antibodies to cellularantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811. Semisolid,gelling, soft-gel, or other formulations (including controlled release)can be used, e.g., when administration to a surgical site is desired.Methods of making such formulations are known in the art and can includethe use of biodegradable, biocompatible polymers. See, e.g., Sawyer etal., Yale J Biol Med. 2006 December; 79(3-4): 141-152.

The pharmaceutical compositions can be included in a container, kit,pack, or dispenser together with instructions for administration.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1. Reducing IL-1 Expression by CRISPR Gene-Engineering in aMouse Model of Osteoarthritis

Sixty C57B mice are selected and distributed into four groups of fifteenmice each. The DMM surgical method is used to induce OA in each of themice. Once the mice have developed OA, the mice are treated as follows:

Group 1: Direct injection into the OA joint a CRISPR AAV vectorengineered to target IL-1α and IL-1β, and silence or reduce theexpression of IL-1 protein.

Group 2: Direct injection into the OA joint a CRISPR AAV vectorengineered with a “nonsense” payload that will not affect an IL-1production; a negative control.

Group 3: Direct injection into the OA joint a CRISPR AAV vectorengineered to target IL-1Ra, and silence or reduce the expression ofIL-1Ra protein.

Group 4: Direct injection into the OA joint sterile buffered saline; acontrol for the injection process.

The mice are monitored before and after treatment to assess effects ontheir locomotion, and exploratory activities. Mechanical sensitivity andchanges to the gait are also monitored. Allodynia and hind limb gripforce may also be monitored.

After about eight weeks, the animals are sacrificed and the OA jointtissue assessed for gross histopathology, and IL-1 expression by IHC.Biomarkers of inflammation are also assessed, for example, MMP-3expression in the OA joint.

Group 1 mice, treated with a CRISPR AAV vector engineered to targetIL-1α and IL-1β, and silence or reduce the expression of IL-1 protein,will show reduced levels of IL-1 by IHC, tissue regeneration byhistopathology, and lower levels of inflammation biomarkers than any ofthe three other Groups. Group 3 mice will show relatively higher levelsof inflammation biomarker than any of the other three groups.

Example 2. Assessing Guide Cutting Efficiency Against Mouse IL1A andIL1B

In Vitro Cleavage Assay

CRISPR guide RNA's (Phosphorothionate-modified sgRNA, Table 3) weredesigned against Exon 4 of Il1a and Exon 4 of Il1b(Il1a-201ENSMUST00000028882.1 and Il1b-201 ENSMUST00000028881.13; see Table 2 fortarget sequences on Exon 4 of Il1a and Exon 4 of Il1b). C57BL/6 mousegenomic DNA was used to amplify Exon 4 of Il1a and Il1b by PCR (PhusionHigh-Fidelity DNA polymerase, NEB cat #M0530S) Il1a primer fwd:CATTGGGAGGATGCTTAGGA, Il1a primer rev: GGCTGCTTTCTCTCCAACAG, Il1b primerfwd: AGGAAGCCTGTGTCTGGTTG, Il1b primer rev: TGGCATCGTGAGATAAGCTG.Amplicons were PCR purified (QiaQuick PCR purification kit cat #28106).Guide cutting efficiency was determined using an in vitro cleavage assayusing 100 ng purified PCR product, 200 ng modified guide RNA (SigmaAldrich) and 0.5 μg TrueCut Spy Cas9 protein V2 (Invitrogen A36498) or0.5 μg Gene Snipper NLS Sau Cas9 (BioVision Cat #M1281-50-1). The twotypes of Cas9, S. pyogenes Cas9 and S. aureus Cas9, were compared fortheir editing capabilities. A 2% agarose gel was used for a qualitativereadout of the cleavage assay.

Editing Cell Lines

CRISPR guide RNA's (Phosphorothionate-modified sgRNA, Table 2) weredesigned against Exon 4 of Il1a and Exon 4 of Il1b (Il1a-201ENSMUST00000028882.1 and Il1b-201 ENSMUST00000028881.13). Guide RNAcutting efficiency was determined in a pool of J774.2 and NIH3T3 cellsusing Sanger sequencing and Synthego ICE (see, e.g., Inference of CRISPREdits from Sanger Trace Data, Hsiau T, Maures T, Waite K, Yang J et al.biorxiv. 2018, which is incorporated by reference herein for allpurposes), or TIDE (see, e.g., Easy quantitative assessment of genomeediting by sequence trace decomposition, Brinkman E, Chen T, Amendola Mand Van Steensel B. Nucleic Acids res 2014, which is incorporated byreference herein for all purposes) web tools to calculate percentediting. The experiment also compared the efficiency of S. pyogenes Cas9and S. aureus Cas9. The cells were electroporated (Amaxa 4D Nucleofectorunit, Lonza) with 5 μg TrueCut Spy Cas9 protein V2 (Invitrogen A36498)or 5 μg EnGen Sau Cas9 protein (NEB M0654T) with 100 pmol modified guideRNA (Sigma Aldrich). SF nucleofector solution and programme CM139 wasused for J774.2 cells and SG nucleofector solution and programme EN158was used for NIH3T3 cells. A cell pellet was taken 3 days' postelectroporation and gDNA was extracted from each pool (Qiagen, DNeasyblood and tissue kit, 69506). Exon 4 of Il1a or Il1b was amplified inthe appropriate pool by PCR (Phusion High-Fidelity DNA polymerase, NEB,cat #M0530S). Il1a primer fwd: TGGTTTCAGGAAAACCCAAG, Il1a primer rev:GCAGTATGGCCAAGAAAGGA, Il1b primer fwd: AGGAAGCCTGTGTCTGGTTG, Il1b primerrev: CTGGGCAAGAACATTGGATT. Amplicons were subjected to Sangersequencing, and analyzed using either the Synthego ICE or TIDE web toolsto determine the absence of wild type sequence in each clone and thepresence of indels resulting in a frameshift in the cDNA sequence.

TABLE 2 Target Il1a and Il1b Sequences Guide Identifier ID Gene ExonCas9 Target Sequence 5'-3' PAM SEQ ID sg43 Il1a 4 S. pyogenesGTATCAGCAACGTCAAGCAA CGG NO: 7 SEQ ID sg44 Il1a 4 S. pyogenesCTGCAGGTCATCTTCAGTGA AGG NO: 8 SEQ ID sg45 Il1a 4 S. pyogenesTATCAGCAACGTCAAGCAAC GGG NO: 9 SEQ ID sg46 Il1a 4 S. pyogenesGCCATAGCTTGCATCATAGA AGG NO: 10 SEQ ID sg47 Il1b 4 S. pyogenesCATCAACAAGAGCTTCAGGC AGG NO: 11 SEQ ID sg48 Il1b 4 S. pyogenesTGCTCTCATCAGGACAGCCC AGG NO: 12 SEQ ID sg49 Il1b 4 S. pyogenesGCTCATGTCCTCATCCTGGA AGG NO: 13 SEQ ID sg50 Il1b 4 S. pyogenesCCTCATCCTGGAAGGTCCAC GGG NO: 14 SEQ ID sg51 Il1a 4 S. aureusTTACTCCTTACCTTCCAGATC ATGGGT NO: 15 SEQ ID sg52 Il1a 4 S. aureusGAAACTCAGCCGTCTCTTCTT CAGAAT NO: 16 SEQ ID sg53 Il1a 4 S. aureusCAACTTCACCTTCAAGGAGAG CCGGGT NO: 17 SEQ ID sg54 Il1b 4 S. aureusGTGTCTTTCCCGTGGACCTTC CAGGAT NO: 18 SEQ ID sg55 Il1b 4 S. aureusCACAGCTTCTCCACAGCCACA AGTAGT NO: 19 SEQ ID sg56 Il1b 4 S. aureusGTGCTGCTGCGAGATTTGAAG CTGGAT NO: 20

TABLE 3 CRISPR Guide RNA's. Guide Identifier ID Gene Exon Cas9cRNA Sequence 5'-3' PAM SEQ ID sg43 Il1a 4 S. pyogenesGUAUCAGCAACGUCAAGCAA CGG NO: 21 SEQ ID sg44 Il1a 4 S. pyogenesCUGCAGGUCAUCUUCAGUGA AGG NO: 22 SEQ ID sg45 Il1a 4 S. pyogenesUAUCAGCAACGUCAAGCAAC GGG NO: 23 SEQ ID sg46 Il1a 4 S. pyogenesGCCAUAGCUUGCAUCAUAGA AGG NO: 24 SEQ ID sg47 Il1b 4 S. pyogenesCAUCAACAAGAGCUUCAGGC AGG NO: 25 SEQ ID sg48 Il1b 4 S. pyogenesUGCUCUCAUCAGGACAGCCC AGG NO: 26 SEQ ID sg49 Il1b 4 S. pyogenesGCUCAUGUCCUCAUCCUGGA AGG NO: 27 SEQ ID sg50 Il1b 4 S. pyogenesCCUCAUCCUGGAAGGUCCAC GGG NO: 28 SEQ ID sg51 Il1a 4 S. aureusUUACUCCUUACCUUCCAGAUC ATGGGT NO: 29 SEQ ID sg52 Il1a 4 S. aureusGAAACUCAGCCGUCUCUUCUU CAGAAT NO: 30 SEQ ID sg53 Il1a 4 S. aureusCAACUUCACCUUCAAGGAGAG CCGGGT NO: 31 SEQ ID sg54 Il1b 4 S. aureusGUGUCUUUCCCGUGGACCUUC CAGGAT NO: 32 SEQ ID sg55 Il1b 4 S. aureusCACAGCUUCUCCACAGCCACA AGTAGT NO: 33 SEQ ID sg56 Il1b 4 S. aureusGUGCUGCUGCGAGAUUUGAAG CTGGAT NO: 34Each cRNA (see, e.g., Table 3) was synthesized as a single guide RNAconsisting of the cRNA sequences above fused to the tracrRNA sequencesbelow (see, e.g., SEQ ID Nos: 35-36). In certain embodiments, an A< >Uflip is used to increase guide RNA activity.

Sau Cas9: (SEQ ID NO: 35)GUUAUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUU Spy Cas9: (SEQ ID NO: 36)GUUAUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU

In Vitro Cleavage Assay

FIG. 1A illustrates agarose gel electrophoresis analysis of 100 ng mouseDNA, cleaved by 0.5 μg Spy Cas9 and 200 ng modified guide RNA's 43-46for Il1a gene and 47-50 for IL1B. DNA is cut at a specific site by thecas9 using the guide RNA to create a predictable band pattern on theagarose gel compared to the uncut control (without wishing to be boundby any particular theory, the agarose gel electrophoresis for sg8*appears to show a failed synthesis).

FIG. 1B illustrates agarose gel electrophoresis analysis of 100 ng mouseDNA, cleaved by 0.5 μg Sau Cas9 and 200 ng modified guide RNA's 51-53for Il1a gene and 54-56 for Il1b. DNA is cut at a specific site by theCas9 using the guide RNA to create a predictable band pattern on theagarose gel compared to the uncut control.

Editing Cell Lines

Genomic DNA was extracted from the edited pools and the Il1a or Il1bexon 4 was PCR amplified in the appropriate pools. The PCR products weresent for sanger sequencing and then deconvoluted using TIDE or SynthegoICE software. Synthego ICE was used to deconvolute the Spy Cas9 pools.The software can determine the patterns of editing in each pool based onthe guide RNA sequence and PAM site. It can distinguish between editingwhich has caused an in frame deletion that could lead to a truncatedfunctional protein, and editing which has causes a frameshift mutationwhich will lead to a true knockout. The SauCas9 pools were analysed withTIDE because Synthego ICE software cannot deconvolute SauCas9 editing.TIDE analysis works in a similar way to ICE by determining patterns ofediting in a pool based on the guide RNA and PAM site. However, ratherthan giving a true knockout score, it gives an editing efficiency score,which cannot distinguish between in frame and frameshift editingpatterns. Therefore, editing efficiency scores may over represent theguide RNA's ability to knockout a protein. SpyCas9 is the standardprotein used in CRISPR gene editing. However, it is 4101 bp compared toSau Cas9 which is 3156 bp. Due to the size limitations of packaging someviruses, such as AAV, it was decided to compare the editing capabilitiesof SauCas9 and SpyCas9 to see whether the smaller Sau Cas9 could be usedin the vector being designed for this project.

FIGS. 2A-2D illustrate graphs displaying editing efficiencies of SpyCas9 (FIGS. 2A and 2B) and SauCas9 (FIGS. 2C and 2D) used with a rangeof guide RNA's in J774.2 (“J”) and NIH3T3 (“N”) cells. Editingefficiencies were determined using Synthego ICE or TIDE sangerdeconvolution software. FIG. 2A: knock out efficiency of Il1a usingguide RNA 43-46 with SpyCas9 in J774.2 and NIH3T3. Synthego ICE was usedto deconvolute the sanger sequence trace and determine knock outefficiency. FIG. 2B: knock out efficiency of Il1b using guide RNA 47-50with SpyCas9 in J774.2 and NIH3T3; without wishing to be bound by anyparticular theory, the data for sgRNA8 appears to show a failedsynthesis. Synthego ICE was used to deconvolute the sanger sequencetrace and determine knock out efficiency. FIG. 2C: knock out efficiencyof Il1a using guide RNA 51-53 with saCas9 in J774.2 and NIH3T3. TIDE wasused to deconvolute the sanger sequence trace and determine the editingefficiency. FIG. 2D: knock out efficiency of Il1b using guide RNA 54-56with Sau Cas9 in J774.2 and NIH3T3. TIDE was used to deconvolute thesanger sequence trace and determine the editing efficiency.

Example 3. Reducing IL-1β Expression by CRISPR Gene-Engineering in aMouse Uric Acid Model

Time Course Experiment to Determine Optimal Pre-Treatment Time

A pilot experiment is performed to determine optimal pre-treatment timeof mice with virus prior to challenging the mice with uric acid. Miceare injected with GFP-labeled AAV5 vector into the knee joint. Viralload is then quantified by PCR and location of viral infection isquantified by histology at 3, 5, and 7 days after infection. A treatmenttime that yields robust expression of virus inside the joint is selectedas the optimal lead time for injecting viral vectors into the mice forthe experiments to determine the reduction of IL-1b in a mouse uric acidmodel by a CRISPR AAV vector engineered to target IL-1b and silence orreduce expression of IL-1b.

Experiment to Confirm CRISPR AAV (AAV-spCas9) Knock Down of IL-1bExpression and Treatment Effect in Uric Acid Model

Mice are selected and distributed into three groups:

Group 1: mice injected with a CRISPR AAV vector (AAV-spCas9) engineeredto target IL-1b, and silence or reduce expression of IL-1 protein,

Group 2: mice injected with “scrambled” guide RNA/Cas9 (AAV-spCas9), aCRISPR AAV vector engineered with a payload that will not affect IL-1production, and

Group 3: mice injected with saline.

The mice are then challenged with uric acid after an optimalpre-treatment time. Within 24 hours of injection with uric acid, theanimals are sacrificed and the joint tissue is analyzed for cytokineexpression (e.g., assessed for IL-1 expression by IHC). The joint tissuemay also be assessed for gross histopathology and for expression ofbiomarkers of inflammation.

Group 1 mice treated with a CRISPR AAV vector engineered to targetIL-1b, and silence or reduce the expression of IL-1 protein, will showreduced levels of IL-1 by IHC and lower levels of inflammationbiomarkers than any of the two other groups.

Example 4. Time Course Study of Intra-Articular Injection of AAV in Mice

A study was conducted to evaluate the time course for injecting AAV intothe joint of male C57BL/6 mice.

Materials & Methods

Test Article Identification and Preparation—The eGFP AAVPrime™ PurifiedAdeno-associated Viral Particles: GFP-tagged AAV5 GeneCopoeia™,catalogue No. AB201, lot No. GC08222K1902, 1.18×10¹³ Genome Copies/mL)and AAV6 (GeneCopoeia™, catalogue No. AB401, lot No. GC09242K1905,5.47×10¹² Genome Copies/mL) we supplied. AAV-particles were shipped ondry ice and were stored at −80° C. immediately upon receipt. Just priorto dosing, the AAV-particles were reconstituted in phosphate bufferedsaline (PBS without calcium and magnesium: Corning, lot No. 11419005)for IA dosing at 10 μL per knee. See the study protocol (Appendix A) foradditional details of test article preparation, storage, and handling.

Test System Identification—Male C57BL/6 mice (N=30) that were 8 to 10weeks old were obtained from The Jackson Laboratory (Bar Harbor, Me.).The mice weighed approximately 24 to 29 grams (mean of 26 g) atenrollment on study day 0. The animals were identified by a distinctmark at the base of the tail delineating group and animal number. Afterrandomization, all cages were labeled with protocol number, groupnumbers, and animal numbers with appropriate color-coding (Appendix A).

Environment & Husbandry—Upon arrival, the animals were housed 3 to 5 percage in polycarbonate cages with wood chip bedding and suspended foodand water bottles. The mice were housed either in shoebox cages (staticairflow, approximately 70 in2 floor space) with filter tops or inindividually ventilated pie cages (passive airflow, approximately 70-75in2 floor space). Animal care including room, cage, and equipmentsanitation conformed to the guidelines cited in the Guide for the Careand Use of Laboratory Animals (Guide, 2011) and the applicable BolderBioPATH SOPs.

The animals were acclimated for 4 days prior to being paced in thestudy. An attending veterinarian was on site or on call during the livephase of the study. No concurrent medications were given.

During the acclimation and study periods, the animals were housed in alaboratory environment with temperatures ranging 19° C. to 25° C. andrelative humidity of 30% to 70%. Automatic timers provided 12 hours oflight and 12 hours of dark. The animals were allowed access ad libitumto Envigo Teklad 8640 diet and fresh municipal tap water.

Experimental Design—On study day 0, the mice were randomized by bodyweight into treatment groups. Following randomization, the animals weredosed by intra-articular (IA) injection as indicated in Table 4. Animalbody weights were measured as described in section 8.5.1. The mice wereeuthanized for necropsy and tissue collection at 3 time points (days 3,5, and 7) as described below in the section titled ‘Necropsy Specimens’.

TABLE 4 Group and Treatment Information Dose Dose Conc. Treat- LevelDose (particles/ Dose Group N ment (particles) Vol. ml) Route Regimen 130 GFP- 5 × 10⁹ 10 μL 5 × 10₁₁/mL IA (right 1× tagged knee) (Day 0) AAV5GFP- 5 × 10⁹ 10 μL 5 × 10¹¹/mL IA (left 1× tagged knee) (Day 0) AAV6

Observations, Measurements, and Specimens

Body Weight Measurements—The mice were weighed for randomization onstudy day 0 and again on days 1, 3, 5, and 7. Body weight measurementscan be found in Table 6.

Necropsy Specimens—The mice were necropsied on study days 3, 5, and 7 asindicated in Table 5.

TABLE 5 Necropsy Schedule Group Animal No. Time-point(s) 1  1-10 Day 3 111-20 Day 5 1 21-30 Day 7

At necropsy, the mice were bled to exsanguination via cardiac puncturefollowed by cervical dislocation. Right and left knees were harvestedfrom all animals. The skin and muscle were removed from the joints whilekeeping the joint capsule intact. Joints were flash-frozen separately in15-mL conical tubes labeled with only mouse number, day of collection,and right or left leg. Knee joints were stored frozen at −80° C. forshipment.

Animal Disposition—Animal carcasses were disposed of according to BBPSOPS.

Specimen and Raw Data Storage—Specimens (right and left knee joints),study data, and reports were delivered during or at the completion ofthe study.

Statement of Effect of Deviations on the Quality and Integrity of theStudy—There were no deviations from the study protocol.

Results/Conclusions

On study day 0, male C57BL/6 mice received IA injections of GFP-taggedAAV5 (5×10⁹ particles, 10 μL) into right knees and IA injections ofGFP-tagged AAV6 (5×10⁹ particles, 10 μL) into left knees. The animalswere weighed on study days 0, 1, 3, 5, and 7. Necropsies were performedon study day 3 (animals 1-10), day 5 (animals 11-20), and day 7 (animals21-30), and right and left knee joints were collected for shipment. Thelive portion of this study was completed successfully including animalweighing, dosing, and biological sample collection. All animals survivedto study termination.

REFERENCES

Guide for the Care and Use of Laboratory Animals (8th Edition). NationalResearch Council, National Academy of Sciences, Washington, D C, 2011,which is incorporated by reference herein in its entirety for allpurposes.

TABLE 6 Body Weight and Dose Calculation Data (MTC-UCM-1) TreatmentGroup Group 1 - Day 0 Day 0 Day 1 Day 3 C57Bl/6 Body Dose Body Body Wt.Body IA, Ix Wt. Vol. Wt. % Δ Wt. Body Wt. (D0) (g) 10 ul (ml) (g)Baseline (g) Baseline 1 26.68 0.01 26.53 −0.6% 26.22 −1.7% 2 27.65 0.0127.55 −0.4% 26.54 −4.0% 3 28.64 0.01 27.97 −2.3% 28.57 −0.2% 4 28.130.01 27.60 −1.9% 27.63 −1.8% 5 26.18 0.01 26.07 −0.4% 25.87 −1.2% 626.38 0.01 26.02 −1.4% 26.30 −0.3% 7 29.13 0.01 29.11 −0.1% 28.83 −1.0%8 24.22 0.01 23.90 −1.3% 23.51 −2.9% 9 25.40 0.01 24.97 −1.7% 24.49−3.6% 10 24.85 0.01 24.21 −2.6% 23.86 −4.0% 11 27.76 0.01 27.26 −1.8%28.02 0.9% 12 25.23 0.01 24.77 −1.8% 24.90 −1.3% 13 24.92 0.01 24.45−1.9% 24.59 −1.3% 14 24.33 0.01 24.19 −0.6% 23.86 −1.9% 15 23.82 0.0123.81 0.0% 23.26 −2.4% 16 24.83 0.01 24.39 −1.8% 24.15 −2.7% 17 25.940.01 25.93 0.0% 26.28 1.3% 18 27.21 0.01 27.44 0.8% 27.60 1.4% 19 25.610.01 25.17 −1.7% 25.22 −1.5% 20 27.81 0.01 27.26 −2.0% 26.99 −2.9% 2126.63 0.01 26.63 0.0% 26.71 0.3% 22 26.96 0.01 27.40 1.6% 25.75 −1.5% 2327.69 0.01 27.21 −1.7% 27.22 −1.7% 24 25.90 0.01 25.71 −0.7% 25.45 −1.7%25 24.03 0.01 24.11 0.3% 23.40 −2.6 26 27.60 0.01 26.67 −3.4% 27.00−2.2% 27 27.87 0.01 27.59 −1.0% 27.22 −2.3% 28 24.43 0.01 24.25 −0.7%24.14 −1.2% 29 26.75 0.01 26.02 −2.7% 26.46 −1.1% 30 25.93 0.01 25.990.2% 25.80 −0.5% Mean 26.28 26.01 −1.0% 25.86 −1.6% SE 0.27 0.27 0.2%0.29 0.3% Treatment Group Group 1 - Day 5 Day 7 Change in C57Bl/6 BodyBody Wt Body Body Wt. Body weight IA, Ix Wt. % Δ Wt % Δ from Baseline(D0) (g) Baseline (g) Baseline (g) 1 −0.46 2 −1.11 3 −0.07 4 −0.50 5−0.31 6 −0.08 7 −0.30 8 −0.71 9 −0.91 10 −0.99 11 28.62 3.1% −0.86 1225.04 −0.8% −0.19 13 24.94 0.1% 0.02 14 23.68 −2.7% −0.65 15 23.71 −0.5%−0.11 16 24.26 −2.3% −0.57 17 26.15 0.8% 0.21 18 27.88 2.5% 0.67 1925.64 0.1% 0.03 20 27.71 −0.4% −0.10 21 26.44 −0.7% 26.70 0.3% 0.07 2225.76 −4.5% 25.75 −4.5% −1.21 23 27.53 −0.6% 27.33 −1.3% −0.36 24 25.61−1.1% 26.00 0.4% 0.10 25 23.76 −1.1% 24.10 0.3% 0.07 26 27.44 −0.6%27.18 −1.5% −0.42 27 27.09 −2.8% 27.30 −2.0% −0.57 28 24.50 0.3% 24.761.4% 0.33 29 26.48 −1.0% 26.48 −1.0% −0.27 30 25.73 −0.8% 25.89 −0.2%−0.04 Mean 25.90 −0.6% 26.13 −0.8% −0.25 SE 0.33 0.4% 0.34 0.5% 0.09

Protocol

Test System

Number of animals: 33 (30+3 extra)

Species/Strain or Breed: C57BL/6

Vendor: Jackson

Age/Wt at Arrival: 8-10 weeks old (˜20 grams)

Gender: Male

Age/Wt Range at Study Initiation: At least 9 weeks by study initiation

Acclimation: Will be acclimated for at least 3 days after arrival at BBP

Housing: 3-5 animals/cage

Study Calendar

Mon Tue Wed Thu Fri Sat Sun Week 1 Week 1 Week 1 Week 1 Week 1 Week 1Week 1 Day −4 Day −3 Day −2 Day −1 Day 0 Day 1 Day 2 Distribute Weigh &Weigh animals Random- on arrival ize. into IA groups Injections foraccli- mation Week 2 Week 2 Week 2 Week 2 Week 2 Week 2 Week 2 Day 3 Day4 Day 5 Day 6 Day 7 Day 8 Day 9 Weigh, Weigh, Weigh, Necropsy NecropsyNecropsy Animals Animals Animals 1-10 11-20 21-30

Materials

Name Supplier Cat #* Isoflurane VetOne 502017 Syringes & Needles BD Asneeded Serum Separator Tubes Greiner #450472 (via Fisher) (if needed)Bio-One Li Hep Mini-Collect Greiner #450480 (via Fisher) (if needed)Bio-One EDTA Mini-Collect Greiner #450477 (via Fisher) (if needed)Bio-One K3EDTA (if needed) Covidien #8881311149 (via Fisher) K2EDTAVacutainer BD #367856 (via Fisher) (if needed) Na Hep Vacutainer BD#367871 (via Fisher) (if needed) Li Hep Vacutainer BD #367960 (viaFisher) (if needed)

Test Article and Vehicle Information

Unformulated Test Article Storage Conditions—GFP-tagged AAV5 (Group 1):−80 C; GFP-tagged AAV6 (Group 1): −80° C.

Vehicle Information—GFP-tagged AAV5 (Group 1): PBS (w/o Ca & Mg);GFP-tagged AAV6 (Group 1): PBS (w/o Ca & Mg).

Test Article Formulation Instructions & Calculations—GFP-tagged AAV5(Group 1): Dilute stock to appropriate concentration using PBS;GFP-tagged AAV6 (Group 1): Dilute stock to appropriate concentrationusing PBS.

Dosing Formulations and Vehicle Storage & Stability—GFP-tagged AAV5(Group 1): Dilute just prior to injecting; GFP-tagged AAV6 (Group 1):Dilute just prior to injecting.

Disposition of Test Articles Following Dosing—GFP-tagged AAV5 (Group 1):Discard formulations, retain stock solution for future studies;GFP-tagged AAV6 (Group 1): Discard formulations, retain stock solutionfor future studies.

Live Phase Deliverables

Live Phase Data Collection Type Study Day Grp (An) Details Body WeightDay 0, 1, 3, 5, 7 All (Remaining)

Necropsy Information

Sacrifice Schedule: Group 1 An 1-10: Day 3

-   -   Group 1 An 11-20: Day 5    -   Group 1 An 21-30: Day 7        Method of Euthanasia: Bleed by cardiac puncture to exsanguinate        followed by cervical dislocation.        Time Points: Not Timed

Necropsy Tissue Sample Collection: Storage Type Gr/An Details ConditionDisposition Right Injected All Remove skin Flash Freeze Ship Knee andmuscle (15 ml conical vial*) keeping joint capsule intact Left InjectedAll Remove skin Flash Freeze Ship Knee and muscle (15 ml conical vial*)keeping joint capsule intact *Label tubes with only mouse number, day ofcollection, and left or right leg. Samples will be tested withoutreference to whether they are AAV-2 or AAV-5 injected. Key to beprovided only after PCR completion.

Sample Analysis

Tissue Specimens—Hind limbs from AAV-injected mice were snap-frozen andshipped. On arrival, specimens were transferred to the −80° C. freezerfor storage.

GFP Expression in Target Tissues—Hind limbs (paired) were thawed at roomtemperature and imaged in an IVIS bioluminescence imaging system (LuminaIII; Perkin Elmer). GFP fluorescence was quantified using excitation at488 nm and measuring emission at 510 nm. A total of 4 mice wereevaluated at each time point (3 days, 5 days and 7 days). Tissues fromthe remaining 6 animals at each time point were retained for subsequentconfirmation of viral burden using real-time PCR.

Results—As can be seen in FIG. 3, there was high-level expression of GFPwithin injected knee joints at 3 days post-injection. Viral loadsdecreased slightly at 5 days, then rose again to 7 days. With thelimited sample size in this pilot study there was no significantdifference between the behaviours of AAV-5 and AAV-6.

Discussion—The data from this study support the use of either AAV-5 orAAV-6 for intra-articular delivery of CRISPR-Cas9 into the mouse kneejoint. The levels of both viral serotypes increased from 5 to 7 day,leaving open the possibility that they may have increased further if thefollow-up had been extended to 2 or maybe 3 weeks. Additional work wouldbe needed to confirm this, but the data thus far would suggest thatthere should be an interval of at least one week before the injection ofthe vector and challenge with intra-articular monoiodoacetate (MIA)crystals.

Background & Rationale—The monoiodoacetate (MIA)-induced OA model isused in this work for two reasons. First, natural (spontaneous) OA isextremely uncommon in mice, whereas the injection of MIA results in aninduced model of OA that is relatively fast in onset, predictable andthat provides good clinical correlation to the disease phenotype see inhuman OA patients, including intra-articular inflammation, pain andcartilage degeneration. Second, in contrast with surgical models such asdestabilization of the medial meniscus (DMM) and transection of theanterior cruciate ligament (ACLT), the MIA model does not involvesurgical incision of the joint capsule, making it much more relevant tothe capsules of human patients with OA.

Injection of MIA crystals in rodents reproduces OA-like lesions andfunctional impairment that can be analyzed and quantified by techniquessuch as behavioral testing and objective lameness assessment. MIA is aninhibitor of glyceraldehyde-3-phosphatase and the resulting alterationsin cellular glycolysis eventual cause the death of cells within thejoint, including chondrocytes. Chondrocyte death manifests as cartilagedegeneration and alterations in proteoglycan staining. Mice injectedwith MIA usually exhibit pain-like behavior within 72 hours, andcartilage loss by around 4 weeks post-injection. Increases in IL-1expression have been documented within 2-3 days of injection in rats andin mice.

Study Design—Mice are injected unilaterally with either MIA or thesaline vehicle control (one joint per animal). Within each group, halfof the animals are pre-treated with the AAV-CRISPR-Cas9 vector targetingthe mouse IL-1 beta gene, and the other half are injected with anAAV-CRISPR-Cas9 scrambled control. Animals from both groups will betaken off study at one of two time points: an early time point of 48hours, to allow for assessment of the impact of therapy on the levels ofIL-1 within the synovial fluid, and a late time point of 4 weeks toallow for assessment of the impact of therapy on cartilage breakdown andhistological evidence of osteoarthritis.

Methods

Experimental Animals—A total of 80 mice are used in this study.

The experimental procedures are reviewed and approved by the localIACUC. Mice are housed in micro-isolator cages, fed a standardlaboratory animal diet, and allowed access to water ad libitum.

MIA Model & Anti-IL1 Therapy—Mice are acclimated for a period of 7 daysahead of the study. On the first day of the study, mice areanaesthetized with an inhaled mixture of isoflurane in oxygen. Once asurgical plane of anesthesia has been confirmed, the right hind limb isclipped and the skin scrubbed with a surgical antiseptic. 40 mice(Treated) receive an intra-articular injection of the AAV-CRISPR-Cas9vector targeting IL-1, and the remaining 40 animals (Control) areinjected intra-articularly with the AAV-CRISPR-Cas9 scrambled control.Seven days later, half of the animals in each group are injected in thesame joint with MIA and half with the saline vehicle. This leads to theestablishment of four study groups:

Group 1: Treated-MIA (20 mice)

Group 2: Control-MIA (20 mice)

Group 3: Treated-Vehicle (20 mice)

Group 4: Control-Vehicle (20 mice)

Ten mice per group are euthanized 48 hours after the MIA challenge inorder to document IL-1 levels in the knee joint. The remaining animalswill be housed for 4 weeks in order to evaluate the effects of therapyon pain behavior (behavioral testing, including von Frey testing),lameness (limb use), joint swelling (caliper measurement) and jointpathology (histopathology).

Euthanasia & Tissue Collection—Mice are killed by exsanguination,followed by cervical dislocation. Joints are opened and either flushedfor IL-1 measurement (48-hour group) or immersion fixed in 10% formalinfor decalcified histopathology (4-week group).

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

It is to be understood that the methods described herein are not limitedto the particular methodology, protocols, subjects, and sequencingtechniques described herein and as such can vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the methods and compositions described herein, which will belimited only by the appended claims. While some embodiments of thepresent disclosure have been shown and described herein, it will beobvious to those skilled in the art that such embodiments are providedby way of example only. Numerous variations, changes, and substitutionswill now occur to those skilled in the art without departing from thedisclosure. It should be understood that various alternatives to theembodiments of the disclosure described herein can be employed inpracticing the disclosure. It is intended that the following claimsdefine the scope of the disclosure and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

Several aspects are described with reference to example applications forillustration. Unless otherwise indicated, any embodiment can be combinedwith any other embodiment. It should be understood that numerousspecific details, relationships, and methods are set forth to provide afull understanding of the features described herein. A skilled artisan,however, will readily recognize that the features described herein canbe practiced without one or more of the specific details or with othermethods. The features described herein are not limited by theillustrated ordering of acts or events, as some acts can occur indifferent orders and/or concurrently with other acts or events.Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the features describedherein.

While some embodiments have been shown and described herein, it will beobvious to those skilled in the art that such embodiments are providedby way of example only. It is not intended that the invention be limitedby the specific examples provided within the specification. While theinvention has been described with reference to the aforementionedspecification, the descriptions and illustrations of the embodimentsherein are not meant to be construed in a limiting sense. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention.

Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein can be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

All publications, patents, and patent applications herein areincorporated by reference to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference. In the event ofa conflict between a term herein and a term in an incorporatedreference, the term herein controls.

The invention claimed is:
 1. A pharmaceutical composition for thetreatment or prevention of a joint disease or condition, comprising: atherapeutically effective amount of a recombinant adeno-associated virusof serotype 5 (AAV5) or serotype 6 (AAV6) comprising one or more nucleicacids encoding a Clustered Regularly Interspaced Short PalindromicRepeats (CRISPR) gene-editing system, the system comprising: (i) aCRISPR Associated Protein 9 (Cas9) protein; and (ii) at least one guideRNA targeting an IL-1α gene, wherein: the at least one guide RNAcomprises a crRNA sequence that is complementary to a target sequence inexon 4 of the IL-1α gene, the crRNA sequence forms no nucleotidemismatches with the target sequence, and the target sequence is adjacentto a protospacer adjacent motif (PAM) sequence for the Cas9 protein, andwherein the pharmaceutical composition is capable of reducinginflammation in a joint following injection of the pharmaceuticalcomposition into the joint.
 2. The pharmaceutical composition of claim1, wherein the at least one guide RNA comprises a sequence selected fromthe group consisting of SEQ ID NO: 21-24 and 29-31.
 3. Thepharmaceutical composition of claim 1, wherein the one or more nucleicacids comprise a first nucleic acid encoding (i) the CRISPR AssociatedProtein 9 (Cas9) protein and (ii) the at least one guide RNA.
 4. Thepharmaceutical composition of claim 1, wherein the one or more nucleicacids comprise a plurality of nucleic acids, and wherein the CRISPRAssociated Protein 9 (Cas9) protein and the at least one guide RNA areencoded by different nucleic acids.
 5. The pharmaceutical composition ofclaim 1, wherein the IL-1α gene is a human IL-1α gene.
 6. Thepharmaceutical composition of claim 1, wherein the recombinantadeno-associated virus is of serotype 5 (AAV5).
 7. The pharmaceuticalcomposition of claim 5, wherein the recombinant adeno-associated virusis of serotype 5 (AAV5).
 8. The pharmaceutical composition of claim 1,wherein the recombinant adeno-associated virus is of serotype 6 (AAV6).9. The pharmaceutical composition of claim 5, wherein the recombinantadeno-associated virus is of serotype 6 (AAV6).
 10. A pharmaceuticalcomposition for the treatment or prevention of a joint disease orcondition, comprising: a therapeutically effective amount of arecombinant adeno-associated virus of serotype 5 (AAV5) or serotype 6(AAV6) comprising one or more nucleic acids encoding a ClusteredRegularly Interspaced Short Palindromic Repeats (CRISPR) gene-editingsystem, the system comprising: (i) a CRISPR Associated Protein 9 (Cas9)protein; and (ii) at least one guide RNA targeting an IL-1β gene,wherein: the at least one guide RNA comprises a crRNA sequence that iscomplementary to a target sequence in exon 4 of the IL-1β gene, thecrRNA sequence forms no nucleotide mismatches with the target sequence,and the target sequence is adjacent to a protospacer adjacent motif(PAM) sequence for the Cas9 protein, and wherein the pharmaceuticalcomposition is capable of reducing inflammation in a joint followinginjection of the pharmaceutical composition into the joint.
 11. Thepharmaceutical composition of claim 10, wherein the at least one guideRNA comprises a sequence selected from the group consisting of SEQ IDNO: 25-28 and 32-34.
 12. The pharmaceutical composition of claim 10,wherein the one or more nucleic acids comprise a first nucleic acidencoding (i) the CRISPR Associated Protein 9 (Cas9) protein and (ii) theat least one guide RNA.
 13. The pharmaceutical composition of claim 10,wherein the one or more nucleic acids comprise a plurality of nucleicacids, and wherein the CRISPR Associated Protein 9 (Cas9) protein andthe at least one guide RNA are encoded by different nucleic acids. 14.The pharmaceutical composition of claim 10, wherein the IL-1β gene is ahuman IL-1β gene.
 15. The pharmaceutical composition of claim 10,wherein the recombinant adeno-associated virus is of serotype 5 (AAV5).16. The pharmaceutical composition of claim 14, wherein the recombinantadeno-associated virus is of serotype 5 (AAV5).
 17. The pharmaceuticalcomposition of claim 10, wherein the recombinant adeno-associated virusis of serotype 6 (AAV6).
 18. The pharmaceutical composition of claim 14,wherein the recombinant adeno-associated virus is of serotype 6 (AAV6).